In ovo time-lapse analysis of chick hindbrain neural crest cell migration shows cell interactions during migration to the branchial arches

Hindbrain neural crest cells were labeled with DiI and followed in ovo using a new approach for long-term time-lapse confocal microscopy. In ovo imaging allowed us to visualize neural crest cell migration 2-3 times longer than in whole embryo explant cultures, providing a more complete picture of the dynamics of cell migration from emergence at the dorsal midline to entry into the branchial arches. There were aspects of the in ovo neural crest cell migration patterning which were new and different. Surprisingly, there was contact between neural crest cell migration streams bound for different branchial arches. This cell-cell contact occurred in the region lateral to the otic vesicle, where neural crest cells within the distinct streams diverted from their migration pathways into the branchial arches and instead migrated around the otic vesicle to establish a contact between streams. Some individual neural crest cells did appear to cross between the streams, but there was no widespread mixing. Analysis of individual cell trajectories showed that neural crest cells emerge from all rhombomeres (r) and sort into distinct exiting streams adjacent to the even-numbered rhombomeres. Neural crest cell migration behaviors resembled the wide diversity seen in whole embryo chick explants, including chain-like cell arrangements; however, average in ovo cell speeds are as much as 70% faster. To test to what extent neural crest cells from adjoining rhombomeres mix along migration routes and within the branchial arches, separate groups of premigratory neural crest cells were labeled with DiI or DiD. Results showed that r6 and r7 neural crest cells migrated to the same spatial location within the fourth branchial arch. The diversity of migration behaviors suggests that no single mechanism guides in ovo hindbrain neural crest cell migration into the branchial arches. The cell-cell contact between migration streams and the co-localization of neural crest cells from adjoining rhombomeres within a single branchial arch support the notion that the pattern of hindbrain neural crest cell migration emerges dynamically with cell-cell communication playing an important guidance role

Cell Dynamics During Somite Boundary Formation Revealed by Time-Lapse Analysis

We follow somite segmentation in living chick embryos and find that the shaping process is not a simple periodic slicing of tissue blocks but a much more carefully choreographed separation in which the somite pulls apart from the segmental plate. Cells move across the presumptive somite boundary and violate gene expression boundaries thought to correlate with the site of the somite boundary. Similarly, cells do not appear to be preassigned to a given somite as they leave the node. The results offer a detailed picture of somite shaping and provide a spatiotemporal framework for linking gene expression with cell movements

Segmentation of the vertebrate hindbrain: a time-lapse analysis

The chick hindbrain starts from a simple and relatively uniform axis and becomes segmented into repeating units, called rhombomeres. The rhombomeres become sites of cell differentiation into specific neurons and the location from which neural crest cells emerge from the neural tube to form the peripheral nervous system, which has only been analyzed at distinct time points due to the lack of a method to watch the neural tube as it is shaped into segments. We have developed a whole-embryo explant culture system in order to study cell and tissue movements with time-lapse video microscopy. Quantitative analyses of the neural tube during its segmentation show that not all rhombomeres are shaped by the same mechanism. In the rostral hindbrain, or first three segments, rhombomeres are shaped by an expansion in the lateral width of the mid-rhombomere; either a smaller expansion or a constriction takes place at the rhombomere boundaries. In the caudal hindbrain, the rhombomere boundaries constrict more than the mid-rhombomere lateral widths increase or decrease, leading to the shaping of the segments. Throughout the segmentation process the rostrocaudal lengths of all rhombomeres remain nearly constant indicating that shape changes are influenced by lateral expansions and constrictions of the neural tube

FGF receptor signalling is required to maintain neural progenitors during Hensen's node progression

Previous analyses of labelled clones of cells within the developing nervous system of the mouse have indicated that descendants are initially dispersed rostrocaudally followed by more local proliferation, which is consistent with the progressing node's contributing descendants from a resident population of progenitor cells as it advances caudally. Here we electroporated an expression vector encoding green fluorescent protein into the chicken embryo near Hensen's node to test and confirm the pattern inferred in the mouse. This provides a model in which a proliferative stem zone is maintained in the node by a localized signal; those cells that are displaced out of the stem zone go on to contribute to the growing axis. To test whether fibroblast growth factor (FGF) signalling could be involved in the maintenance of the stem zone, we co-electroporated a dominant-negative FGF receptor with a lineage marker, and found that it markedly alters the elongation of the spinal cord primordium. The results indicate that FGF receptor signalling promotes the continuous development of the posterior nervous system by maintaining presumptive neural progenitors in the region near Hensen's node. This offers a potential explanation for the mixed findings on FGF in the growth and patterning of the embryonic axis

Structure and Emergence of Specific Olfactory Glomeruli in the Mouse

Olfactory sensory neurons (OSNs) expressing a given odorant receptor (OR) gene project their axons to a few specific glomeruli that reside at recognizable locations in the olfactory bulb. Connecting ∼1000 populations of OSNs to the ∼1800 glomeruli of the mouse bulb poses a formidable wiring problem. Additional progress in understanding the mechanisms of neuronal connectivity is dependent on knowing how these axonal pathways are organized and how they form during development. Here we have applied a genetic approach to this problem. We have constructed by gene targeting novel strains of mice in which either all OSNs or those that express a specific OR gene, M72 or M71, also produce green fluorescent protein (GFP) or a fusion of tau with GFP. We visualized OSNs and their axons in whole mounts with two-photon laser scanning microscopy. The main conclusion we draw from the three-dimensional reconstructions is the high degree of morphological variability of mature glomeruli receiving axonal input from OR-expressing OSNs and of the pathways taken by the axons to those glomeruli. We also observe that axons of OR-expressing OSNs do not innervate nearby glomeruli in mature mice. Postnatally, a tangle of axons from M72-expressing OSNs occupies a large surface area of the bulb and coalesces abruptly into a protoglomerulus at a reproducible stage of development. These results differ in several aspects from those reported for the development of glomeruli receiving input from OSNs expressing the P2 OR, suggesting the need for a more systematic examination of OR-specific glomeruli

Mannose binding lectin is required for alphavirus-induced arthritis/myositis

Mosquito-borne alphaviruses such as chikungunya virus and Ross River virus (RRV) are emerging pathogens capable of causing large-scale epidemics of virus-induced arthritis and myositis. The pathology of RRV-induced disease in both humans and mice is associated with induction of the host inflammatory response within the muscle and joints, and prior studies have demonstrated that the host complement system contributes to development of disease. In this study, we have used a mouse model of RRV-induced disease to identify and characterize which complement activation pathways mediate disease progression after infection, and we have identified the mannose binding lectin (MBL) pathway, but not the classical or alternative complement activation pathways, as essential for development of RRV-induced disease. MBL deposition was enhanced in RRV infected muscle tissue from wild type mice and RRV infected MBL deficient mice exhibited reduced disease, tissue damage, and complement deposition compared to wild-type mice. In contrast, mice deficient for key components of the classical or alternative complement activation pathways still developed severe RRV-induced disease. Further characterization of MBL deficient mice demonstrated that similar to C3(-/-) mice, viral replication and inflammatory cell recruitment were equivalent to wild type animals, suggesting that RRV-mediated induction of complement dependent immune pathology is largely MBL dependent. Consistent with these findings, human patients diagnosed with RRV disease had elevated serum MBL levels compared to healthy controls, and MBL levels in the serum and synovial fluid correlated with severity of disease. These findings demonstrate a role for MBL in promoting RRV-induced disease in both mice and humans and suggest that the MBL pathway of complement activation may be an effective target for therapeutic intervention for humans suffering from RRV-induced arthritis and myositis.This work was supported by NIH/NIAMS R01 AR 047190 awarded to MTH

Dimerization of the transmembrane domain of amyloid precursor proteins and familial Alzheimer's disease mutants

<p>Abstract</p> <p>Background</p> <p>Amyloid precursor protein (APP) is enzymatically cleaved by γ-secretase to form two peptide products, either Aβ40 or the more neurotoxic Aβ42. The Aβ42/40 ratio is increased in many cases of familial Alzheimer's disease (FAD). The transmembrane domain (TM) of APP contains the known dimerization motif GXXXA. We have investigated the dimerization of both wild type and FAD mutant APP transmembrane domains.</p> <p>Results</p> <p>Using synthetic peptides derived from the APP-TM domain, we show that this segment is capable of forming stable transmembrane dimers. A model of a dimeric APP-TM domain reveals a putative dimerization interface, and interestingly, majority of FAD mutations in APP are localized to this interface region. We find that FAD-APP mutations destabilize the APP-TM dimer and increase the population of APP peptide monomers.</p> <p>Conclusion</p> <p>The dissociation constants are correlated to both the Aβ42/Aβ40 ratio and the mean age of disease onset in AD patients. We also show that these TM-peptides reduce Aβ production and Aβ42/Aβ40 ratios when added to HEK293 cells overexpressing the Swedish FAD mutation and γ-secretase components, potentially revealing a new class of γ-secretase inhibitors.</p

Multisensor acoustic tracking of fish and seabird behavior around tidal turbine structures in Scotland

Despite rapid development of marine renewable energy, relatively little is known of the immediate and future impacts on the surrounding ecosystems. Quantifying the behavior and distribution of animals around marine renewable energy devices is crucial for understanding, predicting, and potentially mitigating any threats posed by these installations. The Flow and Benthic Ecology 4D (FLOWBEC) autonomous seabed platform integrated an Imagenex multibeam echosounder and a Simrad EK60 multi-frequency echosounder to monitor marine life in a 120◦ sector over ranges up to 50 m, seven to eight times per second. Established target detection algorithms fail within MRE sites, due to high levels of backscatter generated by the turbulent physical dynamics, limiting and biasing analysis to only periods of low current speed. This study presents novel algorithms to extract diving seabirds, fish, and fish schools from the intense backscatter caused by turbulent dynamics in flows of 4ms−1. Filtering, detection, and tracking using a modified nearest neighbor algorithm provide robust tracking of animal behavior using the multibeam echosounder. Independent multifrequency target detection is demonstrated using the EK60 with optimally calculated thresholds, scale-sensitive filters, morphological exclusion, and frequency-response characteristics. This provides sensitive and reliable detection throughout the entire water column and at all flow speeds. Dive profiles, depth preferences, predator–prey interactions, and fish schooling behavior can be analyzed, in conjunction with the hydrodynamic impacts of marine renewable energy devices. Coregistration of targets between the acoustic instruments increases the information available, providing quantitative measures including frequency response from the EK60, and target morphology and behavioral interactions from the multibeam echosounder. The analyses draw on deployments at a tidal energy site in Scotland to compare the presence and absence of renewable energy structures across a range of physical and trophic levels over complete spring-neap tidal cycles. These results can be used to inform how animals forage in these sites and whether individuals face collision risks. This quantitative information can de-risk the licensing process and, with a greater mechanistic understanding at demonstration scales, its predictive power could reduce the monitoring required at future arrays